Fluorescent proteins (FPs) are powerful, specific marker tools for cellular imaging, and their range of applications is continuously expanding (1). Red fluorescent proteins (RFPs) are of particular interest as they extend the color palette for multi-channel and FRET (fluorescence resonance energy transfer) imaging, and the reduced scattering of long-wavelength light makes them attractive as markers for deep-tissue imaging. A performance gap has been noticed for natural RFPs after isolation from marine invertebrates (2-4), in comparison to variants of the classical green fluorescent protein (GFP) (1). In particular, their tendency to form dimers or tetramers can be detrimental for fusion marker applications (4-6). Therefore, an entire “fruit basket” of monomeric FPs in many different hues was engineered from the tetrameric RFP DsRed (1, 7-8). Together with monomeric variants of GFP, eqFP578 and several reef coral proteins, the emission colors of these FPs cover a wide range from blue to red (1, 6, 9-10). Bright far-red fluorescent markers with emission maxima shifted up to 639 nm were developed on the basis of the Entacmaea quadricolor proteins eqFP578 (11).
Important FP applications are in biosensors that report on intracellular conditions via fluorescence resonance energy transfer (FRET) between two or more chromophors (1, 9-10). A large Stokes shift, i.e., a large wavelength separation between excitation and emission peaks assists in channel separation and facilitates the use of FPs in FRET-based applications, in multi-color imaging application and in whole-body and deep tissue imaging. Several red fluorescent proteins with Stokes shifts >47 are characterized by excitation/emission peaks beyond 582/629 nm. In contrast, there is no fluorescent protein known which exhibits a comparably large Stokes shift in the orange red region of the spectrum between 557 and 630 nm.
Accordingly, it could be helpful to provide novel monomeric fluorescent proteins having a comparably large Stokes shift in the orange red region of the spectrum between 557 and 630 nm, in particular to facilitate biological imaging or imaging applications.